Abstract
Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a “dark side” in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for “filling in the void” by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.
Highlights
intravital microscopy (IVM) has emerged as a powerful tool for studying cellular organization and dynamics in intact organisms [1, 2]
We implemented intravital negative contrast imaging by filling the interstitial space with a cell-impermeable fluorescent tracer such as Evans blue and members of the fluorescent dextran family, which provides a fluorescent background to the unlabeled cells when imaged by two-photon microscopy
Negative contrast imaging overcomes a well-known limitation in standard fluorescence microscopy, the fact that one only sees what one labels
Summary
IVM has emerged as a powerful tool for studying cellular organization and dynamics in intact organisms [1, 2]. Advances in IVM have given us the ability to track specific cell populations labeled with fluorescent dyes or fluorescent proteins (FPs). A limitation with specific labeling, is that only cells that are tagged are visualized. As a result, labeled cells often appear isolated, when they are neighbored with many other (unlabeled) cells and extracellular matrix components. This limitation can be partially overcome by incorporating other nonlinear imaging modalities [7], increasing the number of imaging channels and labeling multiple cell populations at the expense of increasing complexity. The method requires a single fluorescence channel, does not entail any modification to existing microscopes, and can be used in tandem with conventional fluorescent tags
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
